1. Field of the Invention
The present invention relates to isolation, identification, synthesis, expression and purification of antibodies reactive with factor IX (FIX)/factor IXa (FIXa) and especially the FIX/FIXa Gla domain. In particular aspects, the invention provides human antibodies reactive with the human FIX/FIXa Gla domain. The invention further relates to compositions, especially pharmaceutical compositions, articles of manufacture, and methods of inhibiting the activation of FIX/FIXa and inhibiting FIX/FIXa dependent coagulation.
2. Description of Related Disclosures
Factor IXa is a vitamin K dependent plasma serine protease that participates in both the intrinsic and extrinsic pathways of blood coagulation. The NH2 terminal 43 amino acids (Gla domain) of factor IXa and its zymogen factor IX contain 12 Gla residues formed by the vitamin K-dependent carboxylation of Glu residues. The Gla domain is followed by two epidermal growth factor (EGF) type domains, followed by a carboxy terminal serine protease domain.
The Gla domain of FIX/FIXa contains important structural determinants for interaction with high affinity binding sites on vascular endothelial cells and platelets (Heimark et al., (1983) Biochem. Biophys. Res. Commun. 111:723–731; Ahmad et al., (1994) Biochem. 33:12048–12055; Ryan et al., (1989) J. Biol. Chem. 264:20283–20287; Toomey et al., (1992) Biochemistry 31:1806–1808; Cheung et al., (1992) J. Bio. Chem. 267:20529–20531; Rawala-Sheikh et al., (1992) Blood 79:398–405; Cheung et al., (1996) Proc. Natl. Acad. Sci. USA 93:11068–11073; Prorok et al., (1996) Int. J. Pept. Prot. Res. 48:281–285; Ahmad et al., (1998) Biochemistry 37:1671–1679). In the presence of Ca++ and Mg++ the FIX/FIXa Gla domain adopts different conformations. Coagulation reactions, such as FIX/FIXa-mediated activation of FX proceed with high efficiency on the surface of activated platelets (Ahmad and Walsh (1994) Trends Cardiovasc. Med., 4:271–277).
Antibodies that bind the FIX/FIXa Gla domain have been shown to inhibit FIX/FIXa function, such as cell binding (Cheung et al., (1996) supra; clotting activity (Sugo et al., (1990) Thromb. Res. 58:603–614) and FIX/FIXa activation by FXI (Sugo et al., (1990) supra; Liebman et al., (1987) J. Bio. Chem. 262:7605–7612). Rabbit and murine antibodies to FIX/FIXa have been shown to bind to the C- and N-terminal region of the Gla domain (Liebman et al., (1993) Eur. J. Biochem. 212:339–345 and Sugo et al., (1990) Thromb. Res. 58:603–614). Antibodies reactive with human FIX/IXa have been shown to inhibit the activation of FIX to FIXa and inhibit coagulation in a FIXa dependent assay (Blackburn et al., (1997) Blood 90:Suppl. 1:424a–425a). Active site inhibited FIXa attenuates thrombosis in vivo (Wong et al., (1997) Thromb. Haemost. 77:1143–1147; Benedict et al., (1991) J. Clin. Invest. 88:1760–1765; Spanier et al., (1998) J. Thoracic Cardiovasc. Surgery 115:1179–1188).